Characterization of Antioxidant and α-Glucosidase Inhibitory Compounds of Cratoxylum formosum ssp. pruniflorum and Optimization of Extraction Condition

Cratoxylum formosum ssp. pruniflorum (Kurz.) Gogel (Guttiferae), called kuding tea, is widely distributed in Southeast Asia. In this study, the constituents and biological activity of C. formosum ssp. pruniflorum were investigated. Extract of its leaves, roots and stems showed antioxidant and α-glucosidase inhibitory activity. Interestingly, comparison of the metabolite profiles of leaves, roots and stems of C. formosum ssp. pruniflorum by LC-MS analysis showed a great difference between the roots and leaves, whereas the roots and stems were quite similar. Purification of the roots and leaves of C. formosum ssp. pruniflorum through various chromatographic techniques resulted in the isolation of 25 compounds. The structures of isolated compounds were elucidated on the basis of spectroscopic analysis as 18 xanthones, 5 flavonoids, a benzophenone and a phenolic compound. Among them, a xanthone (16) and a benzophenone (19) were first reported from nature. Evaluation of biological activity revealed that xanthones had a potent α-glucosidase inhibitory activity, while flavonoids were responsible for the antioxidant activity. To maximize the biological activity, yield and total phenolic content of C. formosum ssp. pruniflorum, extraction conditions such as extraction solvent, time and temperature were optimized using response surface methodology with Box–Behnken Design (BBD). Regression analysis showed a good fit of the experimental data, and the optimal condition was obtained as MeOH concentration in EtOAc, 88.1%; extraction time, 6.02 h; and extraction temperature 60.0 °C. α-Glucosidase inhibitory activity, yield and total phenolic content under the optimal condition were found to be 72.2% inhibition, 10.3% and 163.9 mg GAE/g extract, respectively. These results provide useful information about C. formosum ssp. pruniflorum as functional foods for oxidative stress–related metabolic diseases.


Introduction
Diabetes is one of the most common metabolic diseases worldwide. According to the International Diabetes Federation, 463 million adults had diabetes worldwide as of 2019, with these numbers increasing to 578 million by 2030 and 700 million by 2045. In diabetes, the increased blood glucose level leads to release of glucose into the urine. Diabetes is caused by a malfunction of carbohydrate metabolism due to insufficient or abnormal insulin function. Sustained hyperglycemia progresses to various diabetes complications such as cardiovascular diseases, nephropathy, neuropathy and retinopathy [1][2][3].

Plant Material
The leaves, roots and stems of C. formosum ssp. Pruniflorum,were collected from trees of 3-4 m height at Huongkhe District at Hatinh Province (GPS: 18 •

Analysis of Chemical Profile Using LC-MS/MS
For the LC-HRMS/MS study, an Orbitrap Exploris 120 mass spectrometer was linked to a Vanquish UHPLC and diode array detector. The extracts of the leaves, roots and stems of C. formosum ssp. pruniflorum (0.5 mg/mL) were analyzed by YMC-Triart C18 column (100 × 2.1 mm, 1.9 µm), using a gradient system (H 2 O with 0.1% formic acid-CH 3 CN with 0.1% formic acid, 90:10 to 0:100) with a flow rate of 0.3 mL/min. The column oven was preheated to 30 • C, and the injection volume of samples was set at 5 µL. Orbitrap mass analyzer resolution was set at 60,000 for the whole MS scan and 15,000 for the datadependent MS n scan, and mass detection was performed in the m/z range of 200-2000. Spray voltage of 3.5 kV, vaporizer temperature of 275 • C, ion transfer tube temperature of 320 • C, sheath gas flow rate of 6.4 L/min, aux gas flow rate of 12 L/min, and sweep gas flow rate of 2.2 L/min were the ion source characteristics for HESI. Ion collisions in the Orbitrap detector occurred at a normalized higher-energy collision dissociation (HCD) energy of 30%. The four most intense ions' MS 2 spectra were acquired using MS/MS fragmentation with the data-dependent MS n mode, and a dynamic exclusion filter was used to prevent further fragmentation of the ions within 2.5 s after getting the MS 2 spectrum.

Measurement of Antioxidant and α-Glucosidase Activity
The inhibitory effect on α-glucosidase was measured using α-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20) [30]. A test sample was mixed with 80 µL enzyme buffer and 10 µL α-glucosidase and incubated for 15 min at 37 • C. Then, after the addition of 10 µL p-nitrophenyl α-D-glucopyranoside solution for enzyme reaction, the amount of pnitrophenol that was cleaved by the enzyme was determined by measuring the absorbance at 405 nm in a 96-well microplate reader. Acarbose was used as a positive control. The antioxidant activity was evaluated by measuring the DPPH radical scavenging activity using ascorbic acid as a positive control [30].

Quantitation of Phenolic and Flavonoid Contents
The leaves, roots and stems of C. formosum ssp. pruniflorum were extracted respectively with 80% MeOH. The total amounts of phenolic and flavonoid contents of each extract were quantitated using Folin-Ciocalteu assay and aluminum chloride colorimetirc assay, respectively [31,32].

Extraction and Isolation
For the purification of compounds, the dried powder of C. formosum ssp. pruniflorum root (87.0 g) was extracted with 80% MeOH (1 L × 2) at room temperature. The MeOH extract (4.8 g) was suspended in H 2 O and partitioned successively with CH 2 Cl 2, EtOAc and n-BuOH.

Response Surface Methodology
A Box-Behnken design (BBD) with three variables such as extraction solvent (X 1 ), extraction time (X 2 ) and extraction temperature (X 3 ) was chosen, with the three variables serving as independent variables, and α-glucosidase inhibitory effects together with yield and total phenolic content were determined as the dependent responses. Regression analysis was performed according to the experimental data; the mathematical model can be explained by the following equation: Y is the response, β0 is the constant coefficient, βi are the linear coefficients, βii are the quadratic coefficients and βij are the interaction coefficients. The statistical significance of the coefficients in the regression equation was checked by analysis of variance (ANOVA). The fitness of the polynomial model equation to the responses was evaluated with the coefficients of R 2 .

Comparison of Different Parts of C. formosum ssp. pruniflorum
Plant components are synthesized through plant-specific biosynthetic pathways, so there are similarities throughout the plant. However, if you subdivide it a little more, it shows some differences in constituents for each part of the plant, which leads to a difference in efficacy [32][33][34]. Therefore, we first compared the antioxidant and anti-diabetic efficacy of the parts of this plant, such as leaves, stems and roots. Since xanthone and flavonoid components have been known as major components of this plant, the contents in each part of the plant were also compared.
As shown in Table 1, all the parts of this plant, including the leaves, roots, and stems, showed antioxidant and α-glucosidase inhibitory effects. However, there were differences in the efficacy. The antioxidant effect was observed most strongly in the leaves, and the roots and stems also showed the efficacy. However, the roots showed the most excellent α-glucosidase inhibitory efficacy with an IC 50 value of 2.0 µg/mL, followed by the leaves with an IC 50 value of 3.9 µg/mL, but relatively weak efficacy in the case of the stem. As a result of comparing the contents of components, both flavonoid and phenol contents were highest in leaves. In particular, in the case of leaves, the content of flavonoid was relatively high, whereas phenolic compounds were observed to be high in roots and stems. We further compared the chemical profiles of each part of C. formosum ssp. pruniflorum. As shown in Figure 1, the MS/MS chromatogram of leaves of C. formosum ssp. pruniflorum was quite different from that of roots and stems. Peak analysis by LC-MS/MS showed that mangiferin and quercetin-3-O-glucopyranoside were the major constituents of leaves, whereas α-mangostin, 7-geranyloxy-1,3-dihydroxyxanthone, and cochinchinone A were the major constituents of roots and stems ( Table 2). The chemical patterns of the roots and stems were quite similar, but the components of the roots were more diverse than those of the stems and showed a higher content. Therefore, roots and leaves were selected for further purification of compounds.

Comparison of Different Parts of C. formosum ssp. pruniflorum
Plant components are synthesized through plant-specific biosynthetic pathways, so there are similarities throughout the plant. However, if you subdivide it a little more, it shows some differences in constituents for each part of the plant, which leads to a difference in efficacy [32][33][34]. Therefore, we first compared the antioxidant and anti-diabetic efficacy of the parts of this plant, such as leaves, stems and roots. Since xanthone and flavonoid components have been known as major components of this plant, the contents in each part of the plant were also compared.
As shown in Table 1, all the parts of this plant, including the leaves, roots, and stems, showed antioxidant and α-glucosidase inhibitory effects. However, there were differences in the efficacy. The antioxidant effect was observed most strongly in the leaves, and the roots and stems also showed the efficacy. However, the roots showed the most excellent α-glucosidase inhibitory efficacy with an IC50 value of 2.0 µg/mL, followed by the leaves with an IC50 value of 3.9 µg/mL, but relatively weak efficacy in the case of the stem. As a result of comparing the contents of components, both flavonoid and phenol contents were highest in leaves. In particular, in the case of leaves, the content of flavonoid was relatively high, whereas phenolic compounds were observed to be high in roots and stems. We further compared the chemical profiles of each part of C. formosum ssp. pruniflorum. As shown in Figure 1, the MS/MS chromatogram of leaves of C. formosum ssp. pruniflorum was quite different from that of roots and stems. Peak analysis by LC-MS/MS showed that mangiferin and quercetin-3-O-glucopyranoside were the major constituents of leaves, whereas α-mangostin, 7-geranyloxy-1,3-dihydroxyxanthone, and cochinchinone A were the major constituents of roots and stems ( Table 2). The chemical patterns of the roots and stems were quite similar, but the components of the roots were more diverse than those of the stems and showed a higher content. Therefore, roots and leaves were selected for further purification of compounds.

Isolation and Characterization of the Constituents of C. formosum ssp. pruniflorum
Using various chromatography methods, 16 (1-16) and 9 (17-25) compounds were isolated from the roots and leaves of this plant, respectively. The structures of the isolated compounds were identified using spectroscopic methods as 2 new compounds (16 and 19) together with 23 known compounds.  [35]. The presence of the xanthone skeleton was confirmed from the 12 aromatic signals at [δ C 110. The presence of hydroxy group in the geranyl group was suggested by the oxymethine carbon at δ C 69.7 (C-7 ) and two methyl signals at δ H 1.18 (6H, s, CH 3 -8 , CH 3 -9 ), which was confirmed by the HMBC correlations from CH 3 -8 , 9 to C-7 . Therefore, compound 16 was suggested to be a xanthone derivative with prenyl and hydroxygeranyl moieties. The positions of the prenyl and hydroxygeranyl moieties were deduced to C-1 and C-4, respectively, by the correlation from H-1 to C-1 and from H-1 to C-4 in the HMBC spectrum. On the basis of the obtained data, compound 16 was determined as shown and named pruniflonone A. . The HMBC correlations from H-2/6 and H-2 /6 to C-7 (C=O) suggested the presence of a benzophenone skeleton. The position of the glucose was determined to be located at C-3 on the basis of HMBC correlation from the anomeric proton (H-1 ) to C-3. Based on these data, compound 19 was determined as shown in Figure 2 and named pruniflonone B. moiety was also confirmed by an anomeric proton at δH 4.81 (1H, d, J = 7.7 Hz, H-1″) together with the glucosyl carbon signals at [δC 101.9 (C-1″), 73.3 (C-2″), 76.0 (C-3″), 70.6 (C-4″), 74.3 (C-5″), 63.5 (C-6″)]. The HMBC correlations from H-2/6 and H-2′/6′ to C-7 (C=O) suggested the presence of a benzophenone skeleton. The position of the glucose was determined to be located at C-3 on the basis of HMBC correlation from the anomeric proton (H-1″) to C-3. Based on these data, compound 19 was determined as shown in Figure 2 and named pruniflonone B.

Evaluation of Antioxidant and α-Glucosidase Inhibitory Activity
The biological activity of the isolated compounds were evaluated by measuring the DPPH radical scavenging and α-glucosidase inhibitory activity. As described above, compounds isolated from C. formosum ssp. pruniflorum in this study are aromatic compounds and can be subdivided according to the compound skeleton as follows: xanthones (1-18), a benzophenone (19), a simple phenolic (20) and flavonoids (21)(22)(23)(24)(25). These isolated compounds showed good antioxidant and α-glucosidase inhibitory activity but differential efficacy depending on the structures (Figure 3).

Evaluation of Antioxidant and α-Glucosidase Inhibitory Activity
The biological activity of the isolated compounds were evaluated by measuring the DPPH radical scavenging and α-glucosidase inhibitory activity. As described above, compounds isolated from C. formosum ssp. pruniflorum in this study are aromatic compounds and can be subdivided according to the compound skeleton as follows: xanthones (1-18), a benzophenone (19), a simple phenolic (20) and flavonoids (21)(22)(23)(24)(25). These isolated compounds showed good antioxidant and α-glucosidase inhibitory activity but differential efficacy depending on the structures (Figure 3).
Xanthones are more effective in the inhibition of α-glucosidase activity, whereas flavonoids are effective in antioxidant activity. Xanthones inhibited α-glucosidase activity with IC 50 values of <50 µM. However, the addition of a hydroxyl group to prenyl or geranyl groups reduced the efficacy, as observed in 1 and 9. The addition of a sugar moiety also showed negative effects on α-glucosidase inhibition. In the case of antioxidant activity, xanthones 3, 6, 9, 17 and 18 showed more than 50% DPPH radical scavenging activity at 50 µM. Considering the structure, dihydroxy groups are important for the antioxidant activity of xanthones. In the case of flavonoids, flavonoids except compound 23 showed good antioxidant activity. Similar to xanthones, flavonoids with dihydroxy groups exerted antioxidant activity. Related to the α-glucosidase inhibitory activity of flavonoids Xanthones are more effective in the inhibition of α-glucosidase activity, whereas flavonoids are effective in antioxidant activity. Xanthones inhibited α-glucosidase activity with IC50 values of <50 µM. However, the addition of a hydroxyl group to prenyl or geranyl groups reduced the efficacy, as observed in 1 and 9. The addition of a sugar moiety also showed negative effects on α-glucosidase inhibition. In the case of antioxidant activity, xanthones 3, 6, 9, 17 and 18 showed more than 50% DPPH radical scavenging activity at 50 µM. Considering the structure, dihydroxy groups are important for the antioxidant activity of xanthones. In the case of flavonoids, flavonoids except compound 23 showed good antioxidant activity. Similar to xanthones, flavonoids with dihydroxy groups exerted antioxidant activity. Related to the α-glucosidase inhibitory activity of flavonoids of C. formosum ssp. pruniflorum, compound 21 without any sugar moieties showed good inhibition. However, benzophenone (19) exerted a weak effect on both antioxidant and αglucosidase inhibition.
As described in Table 1, the extract of C. formosum ssp. pruniflorum exhibited α-glucosidase inhibitory and antioxidant activity. It contains xanthones, flavonoids and benzophenone, and most of them showed α-glucosidase inhibitory and/or antioxidant activity (Figure 3). In the case of the newly reported compounds in this study, compound 19 showed antioxidant efficacy, but compound 16, unfortunately, had weak efficacy. Conclusively, although the efficacies of compounds were quite different in each compound, xanthones and flavonoids were suggested to contribute to the antioxidant and α-glucosidase inhibitory potentials of C. formosum ssp. pruniflorum.
Differences were also observed depending on plant parts. For the α-glucosidase inhibition, the root extract showed the best activity, whereas the leaf extract showed the strongest antioxidant activity. Investigation of the constituents showed that the roots contained xanthones as major components and the leaves had flavonoids, which were consistent with the HRESI-MS/MS chromatogram (Figure 1). Measurement of biological activities of isolated compounds suggested that xanthone had α-glucosidase inhibitory potential, whereas flavonoids were more effective in antioxidant activity, which supported differential efficacy of the extract for each part.
Taken together, these results suggested the components and efficacy of C. formosum ssp. pruniflorum, which are differential depending on each part and can be used for the development of a marker component of each part.  As described in Table 1, the extract of C. formosum ssp. pruniflorum exhibited αglucosidase inhibitory and antioxidant activity. It contains xanthones, flavonoids and benzophenone, and most of them showed α-glucosidase inhibitory and/or antioxidant activity (Figure 3). In the case of the newly reported compounds in this study, compound 19 showed antioxidant efficacy, but compound 16, unfortunately, had weak efficacy. Conclusively, although the efficacies of compounds were quite different in each compound, xanthones and flavonoids were suggested to contribute to the antioxidant and α-glucosidase inhibitory potentials of C. formosum ssp. pruniflorum.
Differences were also observed depending on plant parts. For the α-glucosidase inhibition, the root extract showed the best activity, whereas the leaf extract showed the strongest antioxidant activity. Investigation of the constituents showed that the roots contained xanthones as major components and the leaves had flavonoids, which were consistent with the HRESI-MS/MS chromatogram (Figure 1). Measurement of biological activities of isolated compounds suggested that xanthone had α-glucosidase inhibitory potential, whereas flavonoids were more effective in antioxidant activity, which supported differential efficacy of the extract for each part.
Taken together, these results suggested the components and efficacy of C. formosum ssp. pruniflorum, which are differential depending on each part and can be used for the development of a marker component of each part.

Optimization of Extraction Conditions Using Response Surface Metholodogy
The roots of C. formosum ssp. pruniflorum showed strong α-glucosidase inhibitory effects, and xanthones were assigned as active compounds. The content of active constituents in extract is highly affected by extraction conditions such as extraction solvent, extraction time and extraction temperature, which resulted in the difference in their biological activity [57,58]. Therefore, we further optimized the extraction conditions for maximum α-glucosidase inhibitory effects. Response surface methodology (RSM) is a statistical tool that takes several factors into account simultaneously using rationally designed experiments. The optimal condition can be derived effectively, especially in the case of several variables [59,60]. Therefore, RSM using a Box-Behnken design (BBD) was chosen for the optimization of extraction conditions of C. formosum ssp. pruniflorum for maximum efficiency.
Three variables such as extraction solvent (X 1 ), extraction time (X 2 ) and extraction temperature (X 3 ) were chosen as independent variables, and the range of each variable was determined in the preliminary study. α-Glucosidase inhibitory effects together with yield and total phenolic content were determined as the dependent responses. The variables were coded at three levels (−1, 0 and 1), and the complete design consisted of 15 experimental points including three replications of the center points whose variables were all coded as zero (Table 3). Multiple regression analysis of the experiment data yielded the following second-order polynomial regression equation: Total phenolic content = 163.28 + 33.00X 1 − 2.15X 2 + 3.88X 3 − 46.44X 1 2 + 2.38X 2 2 − 6.11X 3 2 + 1.50X 1 X 2 + 6.38X 1 X 3 − 0.95X 2 X 3. The values of the coefficient determination (R2) and the adjusted coefficient determination (adj. R2) of the predicted model in this response suggested that the regression equation can explain the observed value to a high degree. Insignificant p-values of lack of fit (>0.05) for three responses also indicated the adaptability of this analysis (Table 4).
Among extraction variables, the linear term (X 1 ) of MeOH concentration showed the most significant effect on all three responses. Relationships between the two variables in each response were also shown in a three-dimensional response surface (Figure 4). Consistent with multiple regression analysis, extraction solvent showed the strongest effect on yield, phenolic content and α-glucosidase inhibition (Figure 4A,D,G). Yield was increased with increasing MeOH concentration, but phenolic content and α-glucosidase inhibition were decreased with a continuing increase in MeOH concentration. On fixed temperature at 40 • C, yield was also affected by extraction time (Figure 4B), whereas total phenolic content was affected by extraction temperature when extracted with the mixture of MeOH-EtOAc (1:1) ( Figure 4E). However, compared with extraction solvent, α-glucosidase inhibition showed slight changes as extraction time and temperature changed. Based on these results, the extraction condition for maximum yield, α-glucosidase inhibitory effects and total phenolic content was optimized. The extract prepared using the optimized extraction condition was found to exert 73.9% α-glucosidase inhibitory effects at 1 µg/mL with a yield of 10.9% and a total phenolic content of 163.9 mg GAE/g extract ( Table 5). The total phenolic content in the extract prepared using 15 different extraction conditions showed good correlation with α-glucosidase inhibitory effects, which is consistent with our present study about α-glucosidase inhibitory xanthones.  Based on these results, the extraction condition for maximum yield, α-glucosidase inhibitory effects and total phenolic content was optimized. The extract prepared using the optimized extraction condition was found to exert 73.9% α-glucosidase inhibitory effects at 1 µg/mL with a yield of 10.9% and a total phenolic content of 163.9 mg GAE/g extract ( Table 5). The total phenolic content in the extract prepared using 15 different extraction conditions showed good correlation with α-glucosidase inhibitory effects, which is consistent with our present study about α-glucosidase inhibitory xanthones Collectively, the extraction yield and efficacy of C. formosum ssp. pruniflorum vary depending on the extraction conditions, and an extract with excellent efficacy can be efficiently secured through optimization of the extraction conditions. In addition, consistent with the efficacy of the isolated components, which was demonstrated in this study, the phenolic compounds were important for the efficacy of this plant and can be used as reference components for future product development. Collectively, the extraction yield and efficacy of C. formosum ssp. pruniflorum vary depending on the extraction conditions, and an extract with excellent efficacy can be efficiently secured through optimization of the extraction conditions. In addition, consistent with the efficacy of the isolated components, which was demonstrated in this study, the phenolic compounds were important for the efficacy of this plant and can be used as reference components for future product development.

Conclusions
Comparison of the roots, stems and leaves of C. formosum ssp. pruniflorum showed differences in the chemical profiles and biological activity. An investigation of C. formosum ssp. pruniflorum led to the isolation of 25 phenolic compounds, including 2 new compounds. The structures of the isolated compounds were determined to be xanthones, benzophenone, flavonoids and phenol. Two new compounds were defined as pruniflonone A (16) and pruniflonone B (19). The isolated compounds showed good antioxidant and α-glucosidase inhibitory activity with differences in activity depending on the structures. Optimization of extraction conditions was also studied using RSM for maximum efficacy. In conclusion, the C. formosum ssp. pruniflorum with antioxidant and α-glucosidase inhibitory activity might be beneficial for glucose-related diseases.